Under-diagnosis of vector-borne diseases among individuals suspected of having Scrub Typhus in South Korea

Due to environmental and ecological changes and suitable habitats, the occurrence of vector-borne diseases is increasing. We investigated the seroprevalence of four major vector-borne pathogens in human patients with febrile illness who were clinically suspected of having Scrub Typhus (ST) caused by Orientia tsutsugamushi. A total of 187 samples (182 patient whole blood and sera samples, including 5 follow-up) were collected. Antibodies to Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, and Bartonella henselae were tested by using indirect immunofluorescence assays. Molecular diagnoses were performed using real-time PCR. Of the 182 cases, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases as other febrile diseases (OFDs). The seroprevalence of A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae was 51.4% (19/37), 10.8% (4/37), 86.5% (32/37), and 10.8% (4/37) among the ST group, and 42.8% (62/145), 10.4% (19/145), 57.7% (105/145), and 15.9% (29/145) among the OFD group, respectively. There were no significant differences in the seroprevalence between the ST and the OFD groups. Considering the co-occurrence, 89.0% (162/182) had at least one antibody to tick-borne pathogens, 37.0% (60/162) were positive for two pathogens, 17.3% (28/162) for three pathogens, and 6.2% (10/162) for four pathogens. In real-time PCR, O. tsutsugamushi was positive in 16 cases [15 (40.5%) in ST group and 1 (2.2%) in OFD group], and the four other pathogens were negative in all cases except one confirmed as anaplasmosis. In evaluating the five follow-up samples, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Our data highlighted that acute febrile illness and manifestations suggestive of a vector-borne infection must be recognized and further considered for coinfections in clinical practice and the laboratory.


Indirect immunofluorescent assays for antibodies to other vector-borne pathogens
Commercially available IFA test kits containing the positive and negative control reagents were used to analyze the immunoglobulin G (IgG) of anti-Borrelia burgdorferi, anti-Anaplasma phagocytophilum, anti-Bartonella henselae, and anti-Ehrlichia chaffeensis (Fuller Laboratories, Fullerton, CA, USA). All 187 sera were screened at a 1:64 dilution, according to the manufacturer's instructions. We serially diluted the positive controls at ratios of 1:64, 1:128, 1:256, 1:512, and 1:1,024. The negative control and the serial dilutions of the positive control were assayed with the samples in each run. First, the samples were placed on a slide in contact with the substrate and incubated. The slide was then washed in phosphate-buffered saline to remove unbound antibodies. In the second stage, each well was overlaid with a solution of a fluorescein-labeled antibody to human IgG. The antigen-antibody complexes reacted with the anti-human IgG. Each slide was washed, dried, mounted, and interpreted under a fluorescence microscope (Fig 1). The manufacturer recommended the cutoff titer as 1:512; therefore, the fluorescence intensity of the 1:512 diluted positive control was set to the cutoff level to determine a positive test result. Samples with less fluorescence intensity than the 1:512 positive control were interpreted as negative. The fluorescence intensity of the positive samples was compared to the positive controls, and the titers were graded as follows: 1+, the intensity of the 1:512 diluted positive control; 2+, the intensity of the 1:256 positive control; 3+, the intensity of the 1:128 positive control; and 4+, the intensity of the 1:64 positive control. Titers graded as 1+ and 2+ were defined as low titers, and 3+ and 4+ were defined as high titers.

Real-time PCR for vector-borne pathogens
DNA was extracted from all 187 whole blood samples using the QIAamp DNA Mini Kit (QIA-GEN GmbH, Hilden, Germany) according to the manufacturer's instructions. The real-time PCR was performed using commercial kits for vector-borne pathogens: the Power Chek O. tsutsugamushi Real-time PCR Kit, PowerChek Ehrlichia/Anaplasma Real-time PCR Kit, and PowerChek Rickettsia/Borrelia/Bartonella Real-time PCR Kit (Kogene Bio-tech, Seoul, South Korea). Any positive reaction was re-confirmed by additional PCR and sequencing as previously described (S1 Table) [14,[18][19][20][21].

Statistical analysis
The results of the 182 single and first-drawn samples were included in the statistics, and the 5 follow-up samples from 5 patients were not included. Fisher's exact test or a chi-square test was used to compare categorical variables. Student's t-test was used to compare the continuous variables. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed using either SPSS 27.0 (IBM Corp., Armonk, NY, USA) or the diagnostic test evaluation calculator (MedCalc, Ostend, Belgium).

Ethics statement
The collection of samples for this study was conducted in accordance with the guidelines and approval of the Institutional Review Board of Chonnam National University Hospital (approval no. CNUH-2020-117). A waiver of consent was granted by given the nature of the project dealing with the remaining samples, and no information was used that could lead to patient identification.

Demographics and clinical characteristics
Overall, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases were designated as OFD group (Table 1). The OFD group comprised

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Under-diagnosis of Vector-borne Diseases

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Under-diagnosis of Vector-borne Diseases gastrointestinal diseases (n = 33), respiratory infections (n = 24), central nervous system diseases (n = 17), allergic diseases (n = 17), heart disease (n = 9), malignancy (n = 8), leptospirosis (n = 2), hemorrhagic fever with renal syndrome (n = 2), anaplasmosis (n = 2), severe fever with thrombocytopenia syndrome virus (SFTS) (n = 1) and undifferentiated fever (n = 30). The median age of the ST group was significantly higher than the OFD group (73.1 vs. 64.0 years, P = 0.018), and female predominance was found in the ST group (67.6% vs. 44.8%, P = 0.017). In addition, fever, tick-bite history or eschar, and a history of outdoor activity were more frequent in the ST group. Differences in the laboratory parameters were not significant between the ST and OFD groups, except for in the % of lymphocytes and % of basophils.

Seroprevalence and titers of antibodies to vector-borne pathogens
The  Additionally, the co-occurrence rate of each antibody among the 60.5% (98/162) cases seropositive for multiple pathogens was accessed and is shown in Table 3.

Real-time PCR for vector-borne pathogens
The real-time PCR for O. tsutsugamushi was positive in 16 cases; 12 were antibody-positive cases, and 4 were antibody-negative cases. Real-time PCR assays for E. chaffeensis, Borrelia burgdorferi, B. henselae were all negative, but only one case was positive. This positive reaction for A. phagocytophilum was confirmed by additional sequencing, and the sequence was deposited to GenBank (Accession no. OQ750553) (S1 Fig). This positive sample was derived from the patient who was clinically diagnosed with anaplasmosis by morulae found within granulocytes in the peripheral blood smear (PBS).

Paired follow-up sample data
In addition, we further investigated the paired follow-up samples from five patients (Table 4). They were all clinically diagnosed with ST. In case 1, antibodies for A. phagocytophilum and B. burgdorferi appeared on day 18, which were negative on day 0. In case 2, antibodies for O. tsutsugamushi and A. phagocytophilum appeared. The increase in titer was also observed in case 3. In case 4, the antibodies for O. tsutsugamushi, A. phagocytophilum, and B. henselae turned positive on day 10. In case 5, the antibody for E. chaffeensis appeared on day 2. The follow-up samples demonstrated new antibody appearance or some titer changes, but there were no cases of negative changes in existing antibodies.

Discussion
Tick-and arthropod-borne diseases in humans often share similar clinical features but are epidemiologically and etiologically distinct. In South Korea, it is estimated that more than 10,000 patients are treated every year for tick-or arthropod-borne diseases, such as ST or spotted fever. Despite the clinical relevance of vector-borne infections, in-depth epidemiological studies and research investigations are lacking in Korea. The antibodies to several vector-borne pathogens may overlap because trombiculid mites and ticks share many of the same habitats, and even a single elevated antibody titer can be evidence of exposure to a pathogen, although  [22]. The B. burgdorferi sensu lato (s.l.) complex is a diverse group of worldwide-distributed bacteria, comprised of 21 different species [8]. Eleven species from the B. burgdorferi s.l. complex were identified in and strictly associated with Eurasia and, until now, only B. afzelii, B. garinii and B. valaisiana have been reported in Korea [23][24][25]. Lyme disease, caused by pathogenic members of the B. burgdorferi s.l. complex, typically begins with erythema migrans (˜80%), but *18% of patients have non-specific symptoms such as malaise, fatigue, headache, arthralgias, myalgias, fever, and regional lymphadenopathy without recognition of erythema migrans, for which differential diagnoses are required [26]. Recent Korean data reported 8.1% seroprevalence to B. burgdorferi antibodies among forestry workers in National Park Offices, and they used an in-house IFA method [27]. At the present study, commercially available IFA kits were used, and the high seropositive rate of B. burgdorferi was noticed in both ST and OFD groups, in addition to high titers. Interpreting a single positive serologic result as an infection is challenging, especially in endemic areas [28]. However, in non-endemic areas with low Table 3. The co-occurrence rate of each antibody among the 98 cases positive for multiple (two to four) pathogens. It should also be noted that the co-occurrence of B. burgdorferi with A. phagocytophilum or O. tsutsugamushi antibodies was the most frequently found in this study. Several ecological factors could be considered for these serological results. O. tsutsugamushi is transmitted through bites of trombiculid mites, but not through ticks' bites. However, the co-occurrence of antibodies to several vector-borne bacteria could be predicted since trombiculid mites and ticks transmit B. burgdorferi or Anaplasma spp. and share many of the same habitats [29]. In a previous report, A. phagocytophilum was detected both in Ixodes persulcatus ticks and the blood of humans after tick bites [30]. In addition, several researchers previously announced that Rickettsiae could be transmitted by Ixodes sp. (Rickettsia helvetica and R. monacensis), Haemaphysalis longicornis and Dermacentor marginatus ticks (Rickettsia raoultii), or other ticks [31,32]. Regarding climate change, it is known that warming impacts the activity and aggressiveness of ticks, causing human attacks and the possibility of transmission of severe tickborne pathogens to increase [33]; thus, further caution to infection of various VBDs should be taken as concerns grow in Korea.

Co-occurrence, n (%) Orientia tsutsugamushi Anaplasma phagocytophilum Ehrlichia chaffeensis Borrelia burgdorferi Bartonella henselae
In addition to Borrelia, both A. phagocytophilum and E. chaffeensis were found in H. longicornis and I. persulcatus ticks throughout Korea [34]. Although several seroepidemiological and molecular studies have shown that these agents are present in Asia [35,36], suspecting and diagnosing those infections is not easy. Previous seroprevalence studies showed that 1.8% of serum samples from patients with acute fever were positive for A. phagocytophilum through IFA testing [35]. In 2003, the first Korean case of A. phagocytophilum was detected using molecular and serological methods in Chuncheon, Gangwon [36]. Afterward, Yi et al. found 5 (7.1%) human anaplasmosis cases among 70 Koreans who underwent bone marrow examination due to fever and hemocytopenia. The five anaplasmosis cases were confirmed by PCR, and one of them revealed morulae in the PBS [37]. The detection rate of morulae is known to be 25-75% in the first week of the disease [5,38]. In our study, one of the anaplasmosis cases was also diagnosed by morulae found within granulocytes in the PBS. The patient presented fever, dizziness, and myalgia, and laboratory results showed pancytopenia and increased CRP, AST, ALT, LDH, and BUN. The leptangamushi antibody test was positive for O. tsutsugamushi. The IFA assays were all negative, but the PCR was positive for A. phagocytophilum. The patient samples were taken on the fifth day after symptom onset. In a previous case report, the A. phagocytophilum IFA result on day 5 was negative and the PCR was positive, and the IFA titer began to increase on day 10, whereas the PCR turned negative [39]. The results on day 5 were consistent with our case, but the follow-up sample of this case was not included in this study. Considering the negative O. tsutsugamushi PCR result, it can be inferred that the positive O. tsutsugamushi antibody was caused by the possibility of infection in the past.
In this study, 85.2% of the A. phagocytophilum seropositive group harbored antibodies to Borrelia, indicating presumptive evidence of sharing the same vector. However, only 17.3% of the A. phagocytophilum seropositive group harbored antibodies to E. chaffeensis, and the overall seropositivity to E. chaffeensis and titers were relatively low to other pathogens. Only five patients exhibited high titers of E. chaffeensis. A previous Korean study showed that 1.0% of 1,618 ticks (H. longicornis, I. persulcatus, and I. nipponensis) were E. chaffeensis positive via PCR [40][41][42][43][44], and they suggested the distribution of E. chaffeensis throughout South Korea [25]. Although the seroprevalence of E. chaffeensis may be low in Korea, it is necessary to be cautious in cases with a high titer of E. chaffeensis, which may indicate a high burden of tickborne disease. The presence of the causative agents and potential tick vectors with the capacity to bite humans suggests that the serological data reflect a previously unrecognized but emerging problem in South Korea.
In this study, 87.9% of the B. henselae antibody-positive cases harbored multiple antibodies to vector-borne pathogens. A relatively low titer of B. henselae was observed in the OFD group, whereas a high titer was noticed in the ST group. In Korea, serologic and molecular evidence for B. henselae and B. quintana was observed in ticks and small animals [41,44,45]. According to a previous study, Bartonella DNA was isolated from H. longicornis, H. flava, I. persulcatus, and I. nipponensis [46]. Human infections of B. henselae and B. quintana were also described [47][48][49]. The precise incidence of bartonellosis in Korea has not yet been investigated; however, those reports, including this study, suggest that the burden of bartonellosis in Korea could be higher than expected.
We found the possibility of the coinfection of multiple vector-borne pathogens in febrile illness patients, demonstrating the seropositivity of those pathogens. Primarily, the high titers of antibodies to multiple pathogens support the possibility of co-existence. Follow-up cases also strengthened the possibility of coinfection of ST and other VBDs. Four of the five follow-up ST patients were already seropositive to other vector-borne pathogens, suggesting previous exposure. A previous study of 91 individuals who recovered from ST, the follow-up IgM, IgG, and total Ig positivity rates for 13 years were 37.4% (34/91), 22.0% (20/91), and 76.9% (70/91), respectively [50]. Almost all patients with ST had a frequent outdoor activities history, suggesting that they might be persistently exposed to the risk of tick or mite bites. In follow-up evaluation, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Such changes support the possibility of a coinfection of O. tsutsugamushi and other vector-borne pathogens.
Meanwhile, the positive reaction in IFA assay may be due to the cross-reactive immune responses to vector-borne pathogen-related antigens. They are typically group-specific, although perhaps not species-specific. Previous reports announced that antibodies reactive against E. chaffeensis or A. phagocytophilum could react with other species, impeding epidemiologic distinction between the infections [51]. In our data, a cross-reactive effect might exist in E. chaffeensis, considering that its average titer was very low (1+). The possibility of a crossreaction between the antibodies of those pathogens needs to be further evaluated.
The diagnosis of vector-borne infection generally relies on serologic tests using indirect immunofluorescence assays (IFAs) showing at least a 4-fold increase in the antibody titers between paired sera [52,53]. However, the need for the paired serum samples to be taken over a specific period is the most crucial factor that explains the low effectiveness of IFA tests during the acute phase of the disease. Additionally, to perform the IFA test, conditions such as fluorescent microscopes, dark rooms, and trained laboratory personnel are required. Furthermore, serologic evaluations for vector-borne pathogens other than ST are not usually performed in general laboratories because it is not yet permitted by the Korean Ministry of Food and Drug Safety for clinical diagnosis. Real-time PCR has also been proposed for the early diagnosis of vector-borne infection [54], but buffy coat samples are needed, which require technical expertise for their preparation. The clinical sensitivity of a real-time PCR using serum samples is insufficient and is not commercially available [55].
In the clinical field in South Korea, there is little choice in choosing laboratory tests to diagnose VBDs. IFA assays and PCRs for most vector-borne pathogens are unavailable for routine tests and can only be used for research. Therefore, most clinical diagnoses are restricted to the pathogens only available for routine antibody testing, such as O. tsutsugamushi. Therefore, diagnosing VBDs usually depends on the physician's experience and clinical evidence. With this study, we want to show the possibility of the co-infection of other VBD with O. tsutsugamushi and provoke the recognition of the need for further laboratory evaluation. We suggest that when a patient is suspected of VBD, the IFA tests should be performed for the major pathogens. As shown in our data, the positive rate of PCR is relatively low. Previous reports announced that PCR-negative results do not exclude infection, as the presence in the blood of some vector-borne pathogens can be temporal and transient [56]. In this study, the patient clinically diagnosed with anaplasmosis showed a positive PCR and a negative IFA for Anaplasma. Likewise, molecular diagnosis can be helpful in the acute infection stage when antibodies are at low titers or negative.
Our work had several limitations. First, the titers by the directly diluted test samples were not taken. We indirectly compared the sample fluorescence with the diluted positive control. However, by only counting the fluorescence stronger than that of the 1:256 diluted positive control as positive, the positive result has enough value to suggest the seropositivity. Second, the baseline seroprevalence in healthy controls was not evaluated. A Korean study about ST reported no seroprevalence of IgG with a cutoff value of �1:256 among 216 health checkup personnel [50]. The baseline seroprevalence of the other four vector-borne pathogens is unknown in this geographical region; therefore, a further study of antibody and titer analysis for the four vector-borne pathogens is required. Third, along with the positive IgG antibody result, we did not test the IgM antibody, which could provide further information to distinguish between present and past infections. Fourth, cross-reactions among the pathogens should be excluded using assays such as Western blot. Last, we did not evaluate any samples other than blood samples. Molecular studies using tissue samples, such as eschar, may aid in the determination of the causative agents [57].
Here, we found that the co-existence of vector-borne pathogens in ST and other febrile illnesses may be underestimated. Coinfections should be considered in actual clinical practice and also in the laboratory. Acute febrile illness and manifestations suggestive of vector-borne infection must be recognized and further explored in order to determine the appropriate treatment. Further evaluation methods, such as IFA antibody testing and PCR, are needed to be introduced for routine laboratory work.